Molecular dissecting and targeting YAP1 mediated cancer stemness and immune suppression in advanced gastric adenocarcinoma

项目摘要 这项建议的主要目的是阐明YAP1介导的转移生态位和 免疫抑制在胃腺癌合并腹膜癌病治疗中的应用 发现。GAC是美国和世界范围内的主要健康负担。PC常见，影响约45%的GAC 病人。合并PC的GAC患者生存期短，治疗无效。对分子生物学的理解 个人电脑是有限的。为了应对这一尚未满足的临床挑战，我们建立了个人电脑银行基础设施，旨在 利用这些患者提取的样本来发现和验证我们的新靶点。我们的初步RNAseq 对PC样本的分析显示，包括TIM3和TIM3在内的独特免疫抑制分子有所丰富 表达配体Galectin-9(Gal-9)、转化生长因子-β和VistA，而PD-1/PDL-1和CTLA-4表达较少。YAP1有 与人类发育、谱系可塑性有关，并在许多肿瘤类型中上调。然而，它的 肿瘤微环境(TME)在PC转移和免疫抑制中的作用尚不清楚。 我们的初步数据表明YAP1在GAC的原代和转移性肿瘤细胞中高表达 并与较差的存活率显著相关。基因敲除(KO)YAP1显著降低 小鼠肿瘤干细胞特性、肿瘤形成和PC。此外，肿瘤细胞中YAP1的持续耗尽 增强GAC诱导的CD3和CD8 T细胞应答。通过PC样本的单细胞RNAseq(ScRNAseq) 并通过免疫荧光染色验证，我们注意到YAP1、TIM3配体Gal-9和Dkk1高度 在PC的肿瘤细胞中表达，而TIM3在免疫细胞中表达丰富。来自YAP1HIGH和YAP1 KO的RNAseq 患者来源的肿瘤细胞显示，YAP1缺失后，Gal-9和Dkk1显著减少 而这些因素与患者的生存状况不佳有关。我们假设YAP1高PC细胞是 转移-启动细胞，通过赋予肿瘤细胞肿瘤干细胞属性来协调利基 并通过激活TIM3/Gal-9轴和增加Dkk1旁分泌促进肿瘤免疫抑制 在PC TME中。因此，同时靶向河马/YAP1和免疫检查点(TIM3/Gal-9)可能是一种 增强了战略。为了验证我们的假设，我们提出了三个具体目标：目标1.确定泛函 YAP1在PC干细胞中的表达与肿瘤细胞转移的相关性 体内PDX/PDO模型的建立。目的2.探讨YAP1介导免疫抑制的机制 在PC的TME中。目的3.阐明单独或与TIM3联合抑制YAP1的效果 PDO模型、KP-Luc2同基因小鼠模型、GEMM和正在进行的YAP1临床试验。通过利用病人- ，我们将揭示YAP1介导的PC的功能重要性并通过以下方式阐明其机制 其中YAP1介导的免疫抑制为新的治疗策略。在完成这项研究后，我们 将有很强的理由进行一种新颖的组合，以克服我们在 今天的诊所。

Project Abstract The main objectives of this proposal are to elucidate the mechanisms of YAP1-mediated metastatic niche and immunosuppression in gastric adenocarcinoma (GAC) with peritoneal carcinomatosis (PC) for novel therapeutic discoveries. GAC is a major health burden in the US and worldwide. PC is common affecting ~45% of GAC patients. GAC patients with PC have short survival and treatments are ineffective. Molecular understanding for PC is limited. To address this unmet clinical challenge, we have established PC banking infrastructure aiming to utilize these patient-derived specimens to discover and validate our novel targets. Our preliminary RNAseq profiling of PC specimens revealed an enrichment of unique immune suppressive molecules, including TIM3 and it’s ligand Galectin-9 (Gal-9), TGF-β and VISTA, but less expression of PD-1/PDL-1 and CTLA-4. YAP1 has been implicated in human development, lineage plasticity, and upregulated in many tumor types. However, its role in mediating PC metastases and immune suppression in tumor microenvironment (TME) remain unclear. Our preliminary data suggest that YAP1 is highly expressed in primary and metastatic tumor cells of GAC patients and is significantly associated with poor survival. Genetic knockout (KO) YAP1 significantly decreased cancer stemness traits, tumor formation and PC in mice. Further, depletion of YAP1 in tumor cells consistently increased CD3 and CD8 T cell responses from GAC. Through a single cell RNAseq (scRNAseq) of PC samples and validation using immunofluorescent staining, we noticed that YAP1, TIM3 ligand Gal-9 and DKK1 are highly expressed in tumor cells of PC; while TIM3 is enriched in immune cells. RNAseq from YAP1high and YAP1 KO patient-derived tumor cells revealed that Gal-9 and DKK1 were significantly decreased upon depletion of YAP1 and these factors are associated with poor survival of patients. We hypothesize that YAP1high PC cells are metastasis-initiating cells that orchestrate a niche by conferring cancer stemness attributes to the tumor cells and promote tumor immunosuppression through activating TIM3/Gal-9 axis and increasing paracrine of DKK1 in PC TME. Therefore, simultaneously targeting Hippo/YAP1 and immune checkpoint (TIM3/Gal-9) could be an enhanced strategy. To test our hypothesis, we propose three Specific Aims: Aim 1. Determine the functional relevance of YAP1 in PC stemness and metastases in tumor cells using novel stem cell cloning technology and PDX/PDO models in vivo. Aim 2. To investigate the mechanisms whereby YAP1 mediates immunosuppression in TME of PC. Aim 3. Elucidating efficacy of inhibition of YAP1 alone or in combination with TIM3 inhibition using the PDO models, KP-Luc2 syngeneic mouse model, GEMM, and ongoing YAP1 clinical trial. By utilizing patient- derived PC cells, we will uncover functional importance of YAP1-mediated PC and elucidate the mechanisms by which YAP1 mediated immunosuppression for novel therapeutic strategies. Upon completion of this study, we will have a strong rationale for a novel combination that could overcome the shortcomings we experience in the clinics today.